Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida.

Pectate lyase (PL) depolymerizes pectin and other polygalacturonates (PGAs) and is thought to play a role in bacterial invasion of plants. Production of PL by the soft-rotting pathogen Pseudomonas fluorescens CY091 is regulated by Ca2+. In the presence of Ca2+, this bacterium constitutively synthesizes PL in media containing glucose, glycerol, or PGA and excretes over 87% of total PL into culture fluids. In the absence of Ca2+, the organism fails to use PGA as a carbon source and produces very low levels of PL in media containing glucose or glycerol. Of the small amount of PL produced by the bacterium in Ca(2+)-deficient media, over 78% was detected within the cells, indicating that Ca2+ is critical not only for the production but also for the secretion of PL. The pel gene, encoding an alkaline PL (pI 10.0, Mr 41,000) was cloned and located on the overlapping region of a 4.3-kb SalI and a 7.1-kb EcoRI fragment. The 7.1-kb EcoRI fragment appears to contain a promoter for pel gene expression. A 1.7-kb SalI-XhoI subfragment of the 4.3-kb SalI fragment was cloned into pUC18 to give pROTM2. Escherichia coli cells carrying pROTM2 produce 50 to 100 times more PL than do cells carrying other pectolytic constructs. Production of PL by E. coli (pROTM2) was not affected by carbon sources or by Ca2+. The pI and Mr of PL from E. coli corresponded to values for its counterpart from P. fluorescens. A 0.7-kb BglII-ClaI fragment encoding the pel structural sequence was used to detect pel homologs in various species of fluorescent pseudomonads. Homologous sequences were observed in 10 of 11 strains of P. fluorescens, P. viridiflava, and P. putida. The pel gene in fluorescent pseudomonads is well conserved and may exist and remain repressed in certain strains or species which exhibit nonpectolytic phenotypes under laboratory conditions.     

Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153

A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.     

The role of pectate lyase and the jasmonic acid defense response in Pseudomonas viridiflava virulence

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.     

Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis. The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10. The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.     

Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica

Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

Cloning,expression and characterization of a beta-agarase gene from a marine bacterium,Pseudomonas sp. SK38

A gene (pagA) encoding -agarase from Pseudomonas sp. SK38 was cloned and expressed in Escherichia coli. The structural gene consists of 1011 bp encoding 337 amino acids with a predicted molecular weight of 37326 and has a signal peptide of 18 amino acids. The deduced amino acid sequence showed 57% and 58% homology to -agarase from Pseudoalteromonas atalntica and Aeromonas sp., respectively. The recombinant enzyme was purified and biochemically characterized. The enzyme had maximum activity at pH 9 and 30 °C. It was stable at pHs from 8 to 9 and below 37 °C.     

Molecular cloning and sequencing of a pectate lyase gene from Yersinia pseudotuberculosis.

A pectate lyase gene (pelY) from Yersinia pseudotuberculosis was cloned in Escherichia coli DH-5 alpha. The gene was expressed in either orientation in pUC plasmids, indicating that the insert DNA carried a Y. pseudotuberculosis promoter which functioned in E. coli. However, when cloned in the orientation which placed the coding region downstream of the vector lac promoter, expression of pelY was nine times higher than it was in the opposite orientation and the growth of E. coli cells was inhibited. Nucleotide sequence analysis of the pelY gene disclosed an open reading frame of 1,623 base pairs (PLY). The peptide sequence at the amino-terminal end of the protein contains a typical signal peptide sequence, consistent with the observation that the mature PLY protein accumulated largely in the periplasmic space of E. coli. The pI of PLY produced in E. coli cells was 4.5, and its activity was inhibited 90% or more by EDTA. The enzyme macerated cucumber tissue about 1,000 times less efficiently than did PLe from Erwinia chrysanthemi EC16. The pelY gene has no sequence similarity to the pel genes thus far sequenced from Erwinia spp.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.     

Cloning of a gene for intracellular alginate lyase in a bacterium isolated from a ditch

A DNA fragment with a gene for intracellular alginate lyase in a bacterium A1 isolated from a ditch was cloned using a vector plasmid pKK223-3 and the gene was weakly expressed in Escherichia coli DH1 cells. The alginate lyase produced by E. coli DH1 cells was thought to correspond to A1-I among three kinds of alginate lyases (A1-I, A1-I-1 and A1-I-2) produced by the strain A1. Through this study, CaCl₂ was found to be a useful agent for the screening of microbial alginate lyase-producing colonies on agar plates.     

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica.

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M(r) of 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M(r) and pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.     

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.     

海栖热袍杆菌来源的极耐热碱性果胶裂解酶的表达、纯化及定性

将来源于极端嗜热菌属海栖热袍杆菌Thermotoga maritima MSB8的编码碱性果胶裂解酶的结构基因pelA与新型热激质粒pHsh连接, 得到重组质粒pHsh-pelA, 运用mRNA二级结构预测软件对pHsh-pelA的翻译起始区的二级结构进行优化, 得到了具有最佳mRNA二级结构及自由能的质粒pHsh-pelC。将重组质粒pHsh-pelC转入大肠杆菌JM109(DE3)进行表达, 得到了一种极耐热性碱性果胶裂解酶(PelC)。对重组酶的酶学性质研究发现, 该酶的最适反应温度为90oC, 最适反应pH为8.5, 在pH 8.2~9.8之间酶活力稳定, 95oC酶活半衰期为2 h, 并且该酶依赖Ca2+作为活性离子。在工业生产常用温度60oC下, 该酶能够长时间保持稳定, 并具有较高的酶活力。以多聚半乳糖醛酸(PGA)为底物时, 其动力学参数Km值为0.11 mmol/L, Vmax值为327 U/mg。SDS-PAGE结果显示该重组酶的分子量为43 kD, 与理论值相符。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点, 且重组酶热稳定性非常好, 这对该酶的大规模发酵应用具有重要意义。     

Purification and characterization of an extracellular pectate lyase from an Amycolata sp.

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

辣椒疫霉菌果胶裂解酶PL101基因的克隆及生物信息学分析

【背景】辣椒疫病是一种世界性土传病害,严重影响世界各国辣椒生产,并带来巨大经济损失。果胶裂解酶(pectate lyase,PL)作为一类重要的细胞壁降解酶类是该病的重要致病因子。【目的】对果胶裂解酶基因进行克隆,并对其生物信息学特性进行相关分析,进一步阐明该酶的作用机制。【方法】根据辣椒疫霉菌全基因组序列,以高致病菌株SD33为模板扩增PL101基因的全长cDNA序列,并对其理化性质、跨膜区、亲疏水性、结构域等生物学特性进行分析。【结果】除获得PL101相关生物学特性信息外,还对PL101进行三维结构建模,获得可信度较高的蛋白结构,并确定PL101可能的催化位点为Asp183、Arg212、Arg272三个氨基酸。【结论】对PL101基因的克隆及相关生物学特性的分析为进一步阐明PL功能特性提供参考。